Seizure Sensitivity Is Ameliorated by Targeted Expression of K+–Cl− Cotransporter Function in the Mushroom Body of the Drosophila Brain

Hekmat-Scafe, Daria S.; Mercado, Adriana; Fajilan, Adriel A.; Lee, Ann W.; Hsu, Richard; Mount, David B.; Tanouye, Mark A.

doi:10.1534/genetics.109.109074

Cited by 24 publications

(23 citation statements)

References 62 publications

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“…In kcc DHS1 , a loss of Kcc protein causes seizure-sensitivity [24]; kcc DHS1 seizure-sensitivity can be rescued by supplying Kcc back to neurons or back to glia [25]. Here, we extend these observations of Kcc immunoreactivity with a Kcc-specific antibody (Figure S1) and confocal fluorescence microscopy to show expression is also present in the larval nervous system (Fig.…”

Section: Resultssupporting

confidence: 52%

Modeling Glial Contributions to Seizures and Epileptogenesis: Cation-Chloride Cotransporters in Drosophila melanogaster

2014

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Flies carrying a kcc loss-of-function mutation are more seizure-susceptible than wild-type flies. The kcc gene is the highly conserved Drosophila melanogaster ortholog of K+/Cl− cotransporter genes thought to be expressed in all animal cell types. Here, we examined the spatial and temporal requirements for kcc loss-of-function to modify seizure-susceptibility in flies. Targeted RNA interference (RNAi) of kcc in various sets of neurons was sufficient to induce severe seizure-sensitivity. Interestingly, kcc RNAi in glia was particularly effective in causing seizure-sensitivity. Knockdown of kcc in glia or neurons during development caused a reduction in seizure induction threshold, cell swelling, and brain volume increase in 24–48 hour old adult flies. Third instar larval peripheral nerves were enlarged when kcc RNAi was expressed in neurons or glia. Results suggest that a threshold of K+/Cl− cotransport dysfunction in the nervous system during development is an important determinant of seizure-susceptibility in Drosophila. The findings presented are the first attributing a causative role for glial cation-chloride cotransporters in seizures and epileptogenesis. The importance of elucidating glial cell contributions to seizure disorders and the utility of Drosophila models is discussed.

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Section: Resultssupporting

confidence: 52%

Modeling Glial Contributions to Seizures and Epileptogenesis: Cation-Chloride Cotransporters in Drosophila melanogaster

2014

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“…Despite some lethality later in development, surviving adults bearing the rescue transgene were fertile and could be propagated as a stable stock, indicating that the Rdl MI02957 -Gal4 expression pattern includes all essential Rdl -expressing neurons. Notably, the expression pattern of the Rdl MI02957 -Gal4 line contrasts to the more restricted patterns produced by two previously published promoter-fusion Rdl-Gal4 lines (Hekmat-Scafe et al, 2010; Yuan et al, 2014), neither of which exhibited expression in all previously characterized sites of Rdl expression (Fig. S4).…”

Section: Resultscontrasting

confidence: 55%

Plug-and-Play Genetic Access to Drosophila Cell Types using Exchangeable Exon Cassettes

et al. 2015

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Summary Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here we introduce a simple, versatile method for achieving cell type-specific expression of transgenes that leverages the untapped potential of “coding introns” (i.e. introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted “plug-and-play” cassettes (called “Trojan exons”) that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system.

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“…A developmental increase in the activity of the Cl − -extruding KCC2 relative to Cl − -importing NKCC1 reduces the concentration of [Cl − ] i of neurons to amounts that favor GABA A R-mediated hyperpolarization and inhibition in the mature central nervous system. KCC2 deficiency in worms (93,94), flies (95,96), and mice (97) results in neuronal network hyperexcitability. In humans, pathological KCC2 functional down-regulation and NKCC1 up-regulation, which increases neuronal [Cl − ] i to facilitate GABA A R-mediated depolarization, have been demonstrated or implicated in the pathogenesis of several neurological disorders, including various subtypes of epilepsy, posttraumatic spasticity, neuropathic pain from peripheral nerve injury, and autism (98) (Fig.…”

Section: Inhibition Of Mo25 a Key Spak/osr1 Regulatormentioning

confidence: 99%

The WNK-SPAK/OSR1 pathway: Master regulator of cation-chloride cotransporters

et al. 2014

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The WNK-SPAK/OSR1 kinase complex is composed of the kinases WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase) or the SPAK homolog OSR1 (oxidative stress-responsive kinase 1). The WNK family senses changes in intracellular Cl(-) concentration, extracellular osmolarity, and cell volume and transduces this information to sodium (Na(+)), potassium (K(+)), and chloride (Cl(-)) cotransporters [collectively referred to as CCCs (cation-chloride cotransporters)] and ion channels to maintain cellular and organismal homeostasis and affect cellular morphology and behavior. Several genes encoding proteins in this pathway are mutated in human disease, and the cotransporters are targets of commonly used drugs. WNKs stimulate the kinases SPAK and OSR1, which directly phosphorylate and stimulate Cl(-)-importing, Na(+)-driven CCCs or inhibit the Cl(-)-extruding, K(+)-driven CCCs. These coordinated and reciprocal actions on the CCCs are triggered by an interaction between RFXV/I motifs within the WNKs and CCCs and a conserved carboxyl-terminal docking domain in SPAK and OSR1. This interaction site represents a potentially druggable node that could be more effective than targeting the cotransporters directly. In the kidney, WNK-SPAK/OSR1 inhibition decreases epithelial NaCl reabsorption and K(+) secretion to lower blood pressure while maintaining serum K(+). In neurons, WNK-SPAK/OSR1 inhibition could facilitate Cl(-) extrusion and promote γ-aminobutyric acidergic (GABAergic) inhibition. Such drugs could have efficacy as K(+)-sparing blood pressure-lowering agents in essential hypertension, nonaddictive analgesics in neuropathic pain, and promoters of GABAergic inhibition in diseases associated with neuronal hyperactivity, such as epilepsy, spasticity, neuropathic pain, schizophrenia, and autism.

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Seizure Sensitivity Is Ameliorated by Targeted Expression of K+–Cl− Cotransporter Function in the Mushroom Body of the Drosophila Brain

Cited by 24 publications

References 62 publications

Modeling Glial Contributions to Seizures and Epileptogenesis: Cation-Chloride Cotransporters in Drosophila melanogaster

Modeling Glial Contributions to Seizures and Epileptogenesis: Cation-Chloride Cotransporters in Drosophila melanogaster

Plug-and-Play Genetic Access to Drosophila Cell Types using Exchangeable Exon Cassettes

The WNK-SPAK/OSR1 pathway: Master regulator of cation-chloride cotransporters

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